组织工程化尿路上皮结构体内外构建研究

目的探讨工程化尿路上皮结构的构建方法。方法机械分离获取膀胱黏膜层,胰蛋白酶消化法获取膀胱移行上皮细胞,用DKSFM培养基体外培养扩增。以自制的猪小肠黏膜下层(SIS)为支架材料,将培养的膀胱移行上皮细胞接种到支架材料表面,观察细胞在支架材料表面的生长增殖情况;并将体外构建的细胞-支架材料复合物植入裸鼠背部皮下,不同时间点取材进行组织学和免疫组织化学检测。结果膀胱移行上皮细胞在SIS表面能够黏附、生长和增殖。体外复合培养9d后,膀胱移行上皮细胞铺满SIS表面,呈单层细胞结构。裸鼠体内植入4周和8周后,SIS上种植的膀胱移行上皮细胞形成多层结构,抗细胞角蛋白免疫组化染色,细胞胞浆成棕黄色阳性反应。结论采用组织工程技术能够在体内外初步构建尿路上皮结构,为进一步组织工程泌尿道修复重建实验奠定了基础。

Constructing Tissue-engineered Urothelial Structures in vitro and in vivo

Objective To develop a method for reconstructing urothelial structures using tissue engineering techniques. Methods The bladder mucosa of a puppy dog was mechanically separated, cutted into pieces, and digested with trypsin. The obtained bladder transitional epithelia were cultured with DKSFM and then transplanted to the surface of the small intestinal submucosa(SIS) framework. The compound of the epithelia and small intestinal submucosa(SIS) materials were planted under the back skin of the nude rats. The rats were sacrificed at week 1, 2, 4 and 8 after the transplant. Histological and immunohistochemical examinations were performed to the implanted samples. Results The bladder transitional epithelia adhered to, and grew and proliferated on the surface of the SIS framework. The bladder transitional epithelia covered the entire surface of the framework after nine days of culture in vitro, showing a single-layer cellular structure. The bladder transitional epithelia planted on the SIS framework formed a multi-layer structure after four weeks or eight weeks of transplant to the nude rats. The brown cellular plasma staining was observed, indicating a positive reaction to the immunohistochemical staining with cytokeratin. Conclusion Urothelial structures can be reconstructed in vitro and in vivo with tissue engineering technologies, which lays a technical foundation for further urinary tract reconstructing experiments.